Hemicelluloses and particularly xylan-containing polysaccharides are a valuable source for the production of monosaccharides that can be converted into biofuels, industrial platform chemicals, consumer products, food and feed additives. Due to the heterogeneous chemical structure of this material its degradation requires a series of physicochemical and/or enzymatic treatment steps. Processes that enable an effective and selective hydrolysis of pentose-containing polysaccharides are highly desirable.
An important source of pentoses from biomass is xylan. Xylan constitutes about 15-25% of lignocellulosic biomass and up to 70% of other feedstocks such as oat spealts. Xylans represent one of the major components of plant cell walls and are abundantly found in agricultural waste products, e.g. wheat straw, corn stover, corn cobs, and cotton seed. Xylans consist of xylose monomeric subunits linked by β-1-4-glycosidic bonds in a complex polymer with various other components such as arabinose, glucuronic acid, methylglucuronic acid, and acetyl groups. In cereals, xylans frequently contain side chains of α-1,2- and/or α-1,3-linked L-arabinofuranoside. These substituted xylans are commonly referred to as arabinoxylans. Xylans that are substituted with glucose are referred to as glucoxylans. Also mixed forms of these xylans exist.
Xylanases (β-1,3- or β-1,4-xylan xylohydrolase; E.C. 3.2.1.8) are xylanolytic enzymes that depolymerize xylans, arabinoxylan, and/or other xylose-containing polysaccharides. Endo-xylanases (e.g. endo-β-1,4-xylanase) hydrolyze the internal β-glycosidic linkages in xylan, arabinoxylan, and/or other xylose-containing polysaccharides to produce smaller molecular weight xylo-oligomers or xylose monomers.
Major industrial applications of xylanases today are in the pulp and paper industry to improve the bleachability of pulps and to produce xylose as basis for the sweetener xylitol. Furthermore, xylanases can be used in food and feed compositions which contain cereals (e.g. barley, wheat, maize, rye, triticale, or oats) or cereal by-products that are rich in xylans, arabinoxylans and/or glucoxylans. Addition of xylanases to animal feed or baking products improves the break-down of plant cell walls which leads to better utilization of plant nutrients and/or prolonged bread freshness, respectively. In feed compositions xylanase addition leads to improved animal growth rate and feed conversion. Additionally, the viscosity of feed compositions containing xylan can be reduced by xylanase leading to better acceptability and adsorption.
Despite the relatively high number of known fungal and bacterial xylanases, the number of xylanases which are industrially applicable remains limited. This is mainly due to physical process conditions, such as high temperature and low pH, as well as lack of substrate and/or product selectivity. Such drawbacks limit the use of xylanases.
Typical fungal xylanases are inefficient at temperatures higher than 60° C. and they generate a broad spectrum of sugar products containing mixtures of hexoses and pentoses (Saha, B. C. (2003) Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30:279-291). The lack of product specificity and the rapid deactivation of these enzymes at process temperatures>60° C. limits their application in industrial applications. Higher product specificity and operating temperatures would, however, result in simplified purification procedures and faster product generation, leading to overall process intensification and cost reduction.
While typical fungal xylanase preparations operate between pH 3.5-6.0, they are rapidly deactivated under process conditions outside this pH range (Kulkani, N., Shendye, A., Rao, M. (1999) Molecular and biotechnological aspects of xylanases FEMS Microbiology Reviews 23:411-456; Savitha S, Sadhasivam S, Swaminathan K. Application of Aspergillus fumigatus xylanase for quality improvement of waste paper pulp. Bull Environ Contam Toxicol. 2007 April; 78(3-4):217-21). However, for food and feed applications it is desirable that xylanases are stable and/or operate over a broad pH range.
An additional preferable feature is resistance to proteolytic hydrolysis, which would result in higher process stability. Available xylanase are either not resistant to hydrolysis or lack the desired product specifity or temperature stability (Mendicuti Castro L P, Trejo-Aguilar B A, Aguilar Osorio G. Thermostable xylanases produced at 37 degrees C. and 45 degrees C. by a thermotolerant Aspergillus strain. FEMS Microbiol Lett. 1997 Jan. 1; 146(1):97-102; Li N, Yang P, Wang Y, Luo H, Meng K, Wu N, Fan Y, Yao B. (2008) Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. k11. J Microbiol Biotechnol. 18(3):410-416).
Typically, fungal xylanases have a temperature optimum of about 50° C. and a lower pH optimum than bacterial xylanases. A typical example of a fungal xylanase was isolated from Trichoderma longibrachiatum (Xyl1, Mr: 20 Kda, pl˜5.5, 4). The enzyme has temperature and pH optima around 50° C. and pH 4.5, respectively. The xylanase is stable over a pH range from 3-8 and is deactivated above 55° C. as expected for fungal enzymes. The enzyme preferentially cleaves xylose units from either linear xylan or branched hemicellulose polymers. Like other fungal xylanases it also has a rather broad substrate specificity, leading to significant cellulase and arabinase side activities. Fungal xylanases that have been modified to increase thermostability are disclosed in WO 02/18561A2.
In contrast to fungal xylanase, several xylanases from bacteria have a temperature optimum in the range of 50 to 80° C. One example is a thermostable xylanase from Thermobifida (Thermomonospora) fusca (Mr: 22 kDa, pl˜5) which operates in the pH range between 5-8 and at temperatures of 50-75° C. Other thermostable xylanases are described in WO 03/106654A2
However, for industrial applications highly efficient xylanases with enhanced thermostability are desirable.